1. Field
The present application relates to a laser excitation fluorescent microscope provided with a high-functional dichroic mirror that separates a plurality of types of excitation lights and a plurality of types of fluorescence.
2. Description of the Related Art
When a sample on which multistaining procedure is performed using a plurality of types of fluorescent dyes is observed with a confocal laser scanning fluorescence microscope, a plurality of types of laser lights having different wavelengths are used as excitation lights, and a dichroic mirror for separating the excitation lights and a plurality of types of fluorescence generated in accordance with the excitation lights is used. The dichroic mirror has a wavelength characteristic such that there are a plurality of separation wavelengths (rising points from reflecting bands to transmitting bands). In the present specification, such a dichroic mirror having a plurality of separation wavelengths is referred to as “high-functional dichroic mirror”.
What is shown by a solid line in FIG. 20 is a wavelength characteristic curve of transmittance of a high-functional dichroic mirror disclosed in Non-Patent document 1: Olympus Catalog, Confocal Laser Scanning Microscope FV1000 FLUOVIEW UIS2. Normally, a glass substrate on which a dielectric multilayer is formed is used as the high-functional dichroic mirror. In order to separate a plurality of types of excitation lights and a plurality of types of fluorescence using the dielectric multilayer with high efficiency, it is only required to devise to improve a reflectivity in a reflecting band and a transmittance in a transmitting band, and to suppress a ripple of the wavelength characteristic curve at the time of designing layers of the dielectric multilayer.
However, when a characteristic of a dielectric multilayer is strongly controlled, a total film-thickness of the dielectric multilayer tends to increase. When the total film-thickness is large, a glass substrate is likely to be deformed by a stress of the multilayer, which may distort a shape of a laser spot and lower a spatial resolution of a fluorescence image.
Further, in the wavelength characteristic curve shown in FIG. 20, it is not possible to completely separate the plurality of types of excitation lights and the plurality of types of fluorescence, so that there is a possibility that, for example, a part of the fluorescence generated from the sample is wasted and a detection sensitivity of a fluorescence image is lowered.
Accordingly, the present application has a proposition to provide a highly efficient laser excitation fluorescent microscope.